Natural Killer Cells In Pediatric Acute Lymphoblastic Leukemia Patients At Diagnosis Demonstrate An Inhibitory Phenotype and Reduced Cytolytic Capacity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1397-1397 ◽  
Author(s):  
Rayne H. Rouce ◽  
Takuya Sekine ◽  
Gerrit Weber ◽  
Claude Chew ◽  
Katayoun Rezvani ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Target Cells ◽  
Healthy Controls ◽  
Cytokine Release ◽  
Absolute Count ◽  
Flow Cytometric ◽  
Pediatric All

Abstract Background Natural killer (NK) cells are a key component of innate immunity, with the potential to recognize and kill transformed malignant cells without prior sensitization. A balance between activating and inhibitory signals from cell surface receptors determines NK cell cytotoxicity and cytokine release. Therapeutic approaches to augmenting NK cell function are being explored in various malignancies. Little is known about NK phenotype and function in patients with childhood acute lymphoblastic leukemia (ALL), the most common childhood cancer. Here we describe an inhibitory phenotype and impaired cytolytic function in NK cells from pediatric ALL patients at diagnosis, compared with healthy pediatric controls. Restoring NK function may be a useful therapeutic approach in ALL. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 25 patients with newly diagnosed B-ALL, age 1-16 years, and 7 healthy controls, age 2-13 years, in order to compare NK cell frequency, immunophenotype, and functional activity. NK frequency was assessed by flow cytometric staining for CD56+CD3- cells. NK phenotype was assessed by surface expression of activating receptors NKp30, NKp44, NKp46 and NKG2D and inhibitory receptors KIR2DL1/S1, KIR2DL2/S2, KIR3DL1 and NKG2A. Functional activity was determined by incubation of NKs with target cells, followed by flow cytometric measurement of degranulation (surface CD107a) and cytokine release (intracellular IFNg and TNFa). Targets included the MHC class I deficient K562 cell line and, where available, autologous ALL blasts. Results ALL patients demonstrated significantly lower absolute NK cell counts compared with healthy controls (mean absolute count 168 vs. 406 cells/uL, p = 0.0002). They also exhibited significantly fewer NK cells expressing the activating marker NKp46 (mean absolute count 70 vs. 165, p = 0.016); and a significantly higher percentage of cells expressing the inhibitory marker NKG2A (mean 20.5% vs. 1.95% in controls, p = 0.012) (Fig 1A). In co-culture assays with K562 target cells, ALL patients' NK cells demonstrated inferior degranulation and cytokine release compared to healthy controls (representative data in Fig 1B; mean IFNγ production of 1.2% vs. 4.8%, p = 0.02; mean TNFα production of 1.8% vs. 3.8%, p = 0.06; and mean surface CD107a of 5.4% vs. 15.1%, p = 0.08). ALL samples (n = 3) demonstrated little to no cytokine release when incubated with autologous blasts compared with the response elicited by PMA-ionomycin (representative data in Fig 1C; mean CD107a 0.92% vs. 7.85%, p = 0.04; mean IFNγ 0.26% vs 40.47%, p = 0.10; mean TNFα 0.2% vs 41%, p = 0.008). Conclusion At diagnosis, pediatric ALL patients exhibit a lower frequency of NK cells, an inhibitory phenotype, and decreased cytolytic activity compared to healthy pediatric controls, particularly against autologous leukemic blasts. These results suggest that augmentation of the NK response may be useful therapeutically to improve outcomes in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Artificial Antigen Presenting Cells (aAPC) Expanded NK-Mediated Enhanced Lysis of Allogeneic Tumor Cells Regardless of HLA Class I/KIR Mismatch and Its Implication of Use in Eradicating Acute Lymphoblastic Leukemia (ALL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3023-3023
Author(s):  
Hua Zhang ◽  
Bruce Levine ◽  
Nga Voong ◽  
Alan S. Wayne ◽  
Carl H. June ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Target Cells ◽  
Activating Receptors

Abstract Abstract 3023 Poster Board II-999 NK Killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands play critical roles in maintaining natural killer (NK) cell tolerance, while providing surveillance against pathogens and malignant transformation. Natural killer (NK) cells have been explored as tools for adoptive anti-tumor or leukemia immunotherapy and current models hold that a mismatch or absence of KIR ligands on target cells is essential for efficient NK cell mediated cytolysis. However, new approaches are now available to activate NK cells and the role for KIR mediated signaling in regulating cytotoxicity of activated NK cells has not been well studied. In this study, aAPCs comprising IL15Ra+K562 cells engineered to express 4-1BBL activated and expanded peripheral NK cells in the presence of exogenous IL15 up to 1000-fold in 3 weeks. Compared to resting NK cells, 4-1BBL/IL15-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and showed significantly enhanced cytotoxicity against a multitude of tumor targets including K562, Daudi, Ewing's tumors, osteosarcoma, as well as autologous tumors (50%-90% killing vs. 0%-8% with non-activated NK cells). Meanwhile we could detect little to no influence of KIR signaling in regulating cytotoxicity by aAPC activated NK cells, since sorted CD158a+ and CD158b+ activated NK cells showed similar killing of tumor cells expressing HLA group C1 (CD158b ligand) and/or C2 (CD158a ligand) antigens. In contrast, killer activating receptors (KARs) were indispensable for the cytolysis of solid pediatric tumors by aAPC-activated NK cells, since the killing was significantly inhibited by fusion proteins binding to the ligands of NKG2D, NK p30, p44, p46, p80 (KARs). About 20-40% inhibition of the killing was accomplished when all four activating receptors were blocked, though other activating receptors have not been well defined. Although acute lymphoblastic leukemia (ALL) blasts were refractory to fresh NK cytotoxicity, 4-1BBL/IL15 activated NK cells demonstrated higher lytic activities (20%-50%) against ALL blasts from either patients or cell lines. ALL blast lysis could be completely or partially inhibited by KAR-blocking fusion proteins, indicating that expression levels of KAR ligands vary among ALL cases and other solid tumors. We conclude that KIR ligand mismatch or absence is not essential for effective NK cytotoxicities on either solid tumors or ALL when fully activated NK cells are utilized. This suggests that adoptive therapy with autologous aAPC-activated NK cells may prove effective in some clinical settings, such as ALL, AML, or certain solid tumors. Further studies to assess the impact of KAR ligand expression on aAPC-activated NK killing of ALL blasts are in progress. Percentage of Activated NK Killings vs. Fresh NK's with/without KAR-Ig Fusion Proteins Activated NK (E:T=2.5:1) Fresh NK (E:T=25:1) -KAR-Ig Fc +KAR-Ig Fc SB tumor (Ewing's) 48% 30% 0.5% HOS (Osteo sarcoma) 63% 36% 0.7% Daudi (B. lymphoma) 78% 46% 0.2% REH (ALL) 54% 8% 3% Disclosures No relevant conflicts of interest to declare.

A Novel Method for Propagating Primary Natural Killer (NK) Cells Allows Highly Efficient Expression of Anti-CD19 Chimeric Receptors and Generation of Powerful Cytotoxicity Against NK-Resistant Acute Lymphoblastic Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 306-306
Author(s):  
Chihaya Imai ◽  
Shotaro Iwamoto ◽  
Dario Campana
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Lymphocytes ◽  
Nk Cells ◽  
Treatment Option ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Chimeric Receptors ◽  
Membrane Bound

Abstract Despite intensive chemotherapy, acute lymphoblastic leukemia (ALL) recurs in approximately 20% of children and 65% of adults. For most of these patients, allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment option but risk of relapse after transplantation is high. Donor lymphocyte infusions can suppress leukemia relapse but they are generally ineffective in ALL. We and others have shown that T lymphocytes transduced with anti-CD19 chimeric receptors have remarkable anti-ALL capacity in vitro and in vivo, suggesting the clinical testing of receptor-modified autologous T cells in patients with persistent minimal residual disease. However, the use of allogeneic receptor-modified T lymphocytes after HCT might carry the risk of severe graft-versus-host disease (GvHD). In this setting, the use of CD3-negative NK cells is attractive because they should not cause GvHD. Spontaneous cytotoxicity of NK cells against ALL is weak, if measurable at all. To test whether anti-CD19 chimeric receptors could enhance it, we developed a method to specifically expand human primary NK cells and induce high levels of receptor expression. Specific NK cell expansion has been problematic to achieve with established methods, because CD3+ T cells expand preferentially; even with T-cell depletion, residual T cells typically become prominent after stimulation. We overcame this obstacle by generating a genetically-modified K562 myeloid leukemia cell line that expresses membrane-bound interleukin-15 (IL-15) and 4-1BB ligand (CD137L) (K562-mb15-137L). Peripheral blood mononuclear cells from 8 donors were cultured with K562-mb15-137L in the presence of 10 IU/mL IL-2. After 1 week of culture with K562-mb15-137L, CD3- CD56+ NK cells expanded by 16.3 ± 5.9 fold, whereas CD3+ T cells did not expand. The stimulatory effect of K562-mb15-137L was much higher than that of K562 cells transduced with control vectors, K562 expressing membrane-bound IL-15 or CD137L alone, or K562 expressing wild-type IL-15 instead of membrane-bound IL-15. NK cell exposed to K562-mb15-137L were transduced with a retroviral vector and the anti-CD19-BB-ζ receptor, consisting of the single-chain variable domain of an anti-CD19 monoclonal antibody, the hinge and transmembrane domains of CD8α, and the signaling domains of CD3ζ and 4-1BB. 4-1BB mediates signals that are crucial for immune response to tumors in vivo and significantly improves chimeric receptor signaling. In 27 experiments, mean (± SD) transduction efficiency after 7–14 days was 67.5% ± 16.7%. Seven days after transduction, 92.3% (range 84.7%–99.4%) of cells were CD3- CD56+ NK cells; expression of receptors on the cell surface was high. NK cells expressing anti-CD19-BB-ζ had powerful cytotoxicity against NK-resistant B-lineage ALL cell lines and primary ALL cells. NK cells transduced with anti-CD19-BB-ζ had consistently higher cytotoxicity than those transduced with receptors lacking 4-1BB. The method described here allows specific expansion of primary NK cells and highly efficient transduction of chimeric receptors. Expression of anti-CD19-BB-ζ receptors in NK cells markedly enhances their anti-ALL activity. This approach could be a valuable treatment option for patients with refractory or relapsed B-cell malignancies after HCT.

Pre-Clinical Activity of a Novel FcγRIIIa Engineered CD19 Monoclonal Antibody in B Cell Chronic Lymphocytic Leukemia and Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2351-2351
Author(s):  
Julie M. Roda ◽  
Rosa Lapalombella ◽  
Robert Baiocchi ◽  
Eugene Zhukocsky ◽  
John Desjarlais ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Negative Control ◽  
Lymphocytic Leukemia ◽  
Adcc Activity ◽  
Specific Lysis

Abstract CD19 is a B cell lineage-specific transmembrane signaling protein that controls differentiation and proliferation. CD19 is an attractive therapeutic target due to its high level of expression in numerous B cell malignancies, as well as its lack of expression on non-B cells. Here we report the in vitro anti-tumor activity of a novel humanized monoclonal anti-CD19 Ab (CD19-IgG1, aka XENP5603) and its Fc engineered counterpart (XmAb™CD19, aka XENP5574). XENP5603 induced direct apoptosis in normal CD19+ B cells, but not NK cells, T cells, or monocytes, as determined by flow cytometric staining with annexin V and propidium iodide. XENP5603 also induced significant levels of apoptosis in a number of lymphoblastoid cell lines, including Ramos, Raji, 697, NALM6, and RS4;11 cells. Treatment of primary chronic lymphocytic leukemia (CLL) cells with XENP5603 induced significant cell death in all patients tested (mean, 36% apoptotic cells at 24 hours; range, 13–66%, p < 0.001). Similar apoptosis was noted in cells from a subset of patients (4 of 9) with CD19+ primary acute lymphoblastic leukemia (ALL). Apoptosis of CLL cells treated with XENP5603 was not associated with cleavage of caspase-3, caspase-8, caspase-9, or PARP, but was associated with upregulation of Bim, suggesting a caspase-independent mechanism of cell death. NK cells from normal donors exhibited high levels of ADCC in response to B cell lines coated with XENP5603. Furthermore, NK cells from CLL patients mediated significant ADCC against autologous CLL cells in the presence of XENP5603 (mean, 15% specific lysis at an E:T ratio of 25:1; range, 8–24%; p = 0.04 vs. the negative control Ab). ADCC activity was further increased in the presence of XENP5574, which has the same antigen-recognition sequences as XENP5603 but which contains two mutations in the Fc region that increase FcγRIIIa affinity (mean, 39% specific lysis at an E:T ratio of 25:1; range, 29–51%; p = 0.02 vs. the negative control Ab). ADCC mediated by either CD19 Ab was also significantly higher than that mediated by an equivalent concentration of rituximab (mean, 39% specific lysis with XENP5574 vs. 12% with rituximab; p < 0.001). ADCC in the presence of either Ab was further increased in the presence of the NK cell-activating cytokine IL-2, suggesting that these antibodies might be effectively combined with immune stimulatory adjuvants. Furthermore, NK cell ADCC against CLL cells in the presence of CD19 Abs was found to be dependent on perforin/granzyme release, as treatment with 3,4-dichloroisocoumarin (which inhibits granzyme enzymatic activity) or EGTA (which prevents release of cytotoxic vesicles) potently inhibited ADCC activity. Collectively, these studies provide evidence of the autologous innate immune-mediated cytotoxicity and direct apoptotic activity of XENP5603 and XENP5574. In addition, engineering to enhance FcγRIIIa binding enhances autologous ADCC, providing support for further pre-clinical development of XENP5574 in CD19+ malignancies, including CLL and ALL.

The Role of Her2/Neu in ALL: Long Term Clinical Follow-up of Surface-Positive Patients and Induction of NK Cell ADCC by Trastuzumab in Vitro

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1421-1421
Author(s):  
Sebastian P. Haen ◽  
Benjamin J Schmiedel ◽  
Nora Roth ◽  
Christoph Faul ◽  
Robert Möhle ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Additional Risk Factor ◽  
Cytokine Release ◽  
Breast Cancer Patients ◽  
Time To Relapse

Abstract Abstract 1421 In about one third of patients, acute lymphoblastic leukemia (ALL) cells express Her2/neu (p185Her-2). In epithelial malignancies Her2/neu contributes to oncogenic transformation and metastatic potential, and overexpression often is associated with poor prognosis. Previous reports also suggested an inferior prognosis in patients with Her2/neu-positive ALL, but as of now no study has performed survival analyses. In breast cancer patients, targeting of Her2/neu with the monoclonal antibody Trastuzumab results in improved disease outcome, and induction of antibody dependent cellular cytotoxicity (ADCC) and cytokine release of NK cells contributes to the same. Most recently, application of Trastuzumab in patients with relapsed or refractory ALL was shown to yield promising results, but the mechanisms by which Trastuzumab mediates its efficacy have not been studied so far. Here, we studied patient records and flow cytometry data on Her2/neu expression of patients treated for ALL at our hospital between 2000 and 2011 and determined the effect of Trastuzumab and/or Rituximab on NK cell reactivity against ALL blasts in vitro. In 57 patients (37 men, 20 women, median age at diagnosis 42 years, range 17–83 years) data on Her2/neu expression were available. Her2/neu expression was detected in 15/57 patients (26%). Clinical follow up was assessed for all 57 patients. Median follow-up was 44 months (range 0.5–356 months) until death or last follow-up visit. Overall, 60% of the patients had died at the end of follow-up (25 of 42 Her2/neu-negative and 9 of 15 Her2/neu-positive patients, respectively). Median survival times were 43 (Her2/neu-negative, range 0.5–356 months) and 41 months (Her2/neu-positive, range 3.5–138 months; p = 0.81. Kaplan Meyer regression analysis). Also, median time to relapse was comparable in both groups (22.5 months vs. 15.5 months; p = 0.77). In functional analyses with NK cells, incubation of Her2/neu-positive CD20-positive ALL blasts with Trastuzumab or Rituximab comparably induced ADCC and cytokine release of NK cells. Only minor additional effects were observed upon combined application of both antibodies. When blasts from Her2/neu-negative ALL patients were employed (phentotype CD20-positive Her2/neu-negative or CD20-negative Her2/neu-negative), no effect of Trastuzumab was observed, and Rituximab exerted effects solely with blasts from CD20-positive ALL patients. Taken together, in our patient cohort Her2/neu expression on ALL blasts was not associated with time to relapse or overall survival and thus did not appear to be an additional risk factor. Rather, Her2/neu is a promising target for antibody therapy, especially in patients with Her2/neu-positive CD20-negative ALL, as treatment with Trastuzumab is suitable to induce ADCC and cytokine production of NK cells also against Her2/neu-positive CD20-negative ALL blasts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of miR-652-3p and Effect on Apoptosis and Drug Sensitivity in Pediatric Acute Lymphoblastic Leukemia

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Qian Jiang ◽  
Xiaojing Lu ◽  
Pengli Huang ◽  
Chao Gao ◽  
Xiaoxi Zhao ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Drug Sensitivity ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Healthy Controls ◽  
Leukemia Cell Lines ◽  
Pediatric All ◽  
Mirnas Expression ◽  
New Diagnosis

MicroRNAs (miRNAs) expression profiles were screened in plasma samples from pediatric patients with acute lymphoblastic leukemia (ALL) and healthy controls, using qRT-PCR-based TaqMan low-density miRNA arrays. MiR-652-3p (a circulating miRNA) was downregulated in new diagnosis (ND) patients compared with healthy controls. The levels of miR652-3p were restored in complete remission (CR) but were downregulated again in disease relapse (RE). The expression pattern of miR-652-3p was validated in bone marrow (BM) samples from other pediatric ALL patients. MiR-652-3p was significantly upregulated in BM when the patients (n=86) achieved CR, as compared with the matched ND samples (p<0.001). Moreover, the miR-652-3p levels in BM decreased again in two patients at RE. In addition, the lymphoblastic leukemia cell lines Reh and RS4:11 were found to have lower levels of miR-625-3p than the normal B-cell line. Overexpression of miR-652-3p using agomir increased the sensitivity to vincristine and cytarabine (all p<0.05) and promoted apoptosis (both p<0.05) in Reh and RS4:11 cells. In conclusion, the results suggested that a low level of miR-652-3p might be involved in the pathogenesis of pediatric ALL. Overexpression of miR-652-3p might suppress lymphoblastic leukemia cells, promoting apoptosis and increasing sensitivity to chemotherapeutic drugs.

scholarly journals NK Cells as Possible Prognostic Factor in Childhood Acute Lymphoblastic Leukemia

2019 ◽  
Vol 2019 ◽  
pp. 1-4 ◽  
Author(s):  
Agnieszka Mizia-Malarz ◽  
Grażyna Sobol-Milejska
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Prognostic Factor ◽  
Nk Cells ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Percentage ◽  
Group I ◽  
Group Ii ◽  
Cluster Of Differentiation

Deficiency or impaired function natural killer (NK) cells might result in the development of serious infections and promote the development of malignancies. The aim of our study was to assess the prognostic role of NK cell percentage in bone marrow on the day of acute lymphoblastic leukemia (ALL) diagnosis. 84 children (49 males=58%; median age 5 yrs) with ALL were enrolled. The NK cell percentage was assessed using flow cytometry with antibodies against the cluster of differentiation (CD): CD3, CD56, and CD16. We evaluated two groups: group I (NK+), patients with NK cells in the bone marrow (n=74), and group II (NK-), patients without NK cells in the bone marrow (n=10) (cut-off value of negative <1%). In the patients from group I, the prednisone good response on day 8 and the remission on day 15 of treatment were observed significantly more often (p=.01, p=.03). The children from group I had significantly better survival as compared to those from group II (p=.02) (HR 2.59; 95% CI: 1.38-4.85). The presence of NK cells in the bone marrow at diagnosis can be a prognostic factor in children with ALL. The presented results should be the basis for further research.

Enhanced Elimination of 6-Mercaptopurine or Methotrexate-Treated Acute Lymphoblastic Leukemia Cells by Natural Killer Lymphocytes In Vitro: A Potential Mechanism of Action of Maintenance Chemotherapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2128-2128 ◽  
Author(s):  
Abdual H. Siddiqui ◽  
Mohammad Bhuiyan ◽  
Akila Muthukumar ◽  
Steven Buck ◽  
Yaddanapudi Ravindranath ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Drug Exposure ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Target Cells ◽  
Lymphoma Cells ◽  
Nkg2d Ligands ◽  
Reh Cells

Abstract Abstract 2128 Background: Maintenance chemotherapy (MC) is an important component of childhood B-precursor acute lymphoblastic leukemia (ALL) therapy; however, it is not necessary in the treatment of mature B cell neoplasms. The operational mechanisms of MC are not understood. Improvement in immunologic function including near normal levels of natural killer (NK) lymphocytes was reported during ALL MC. We hypothesize that in addition to their direct cytotoxicity, MC drugs alter surviving lymphoblasts, rendering them susceptible to innate immune response, likely through cell mediated cytotoxicity via stress proteins such as NKG2D ligands, co-stimulatory or adhesion molecules. Objective: The effect of 6-mercaptopurine (6MP) or methotrexate (MTX) treatment of B-precursor and mature B leukemia/lymphoma cells in their elimination by NK lymphocytes was investigated in this study. Design and Methods: Allogeneic NK cell-mediated elimination of REH (TEL/AML-positive B-precursor ALL) and Raji (mature B cell lymphoma) cells treated with standard MC drugs was studied. High dose cytarabine (Ara-C) and MTX are used during the consolidation chemotherapy; therefore, Ara-C and MTX-resistant REH and Raji cell sub-lines were established by exposing wild type cells to increasing concentrations of drugs over several months. Natural killer cells from 17 healthy volunteers were separated using the MACS NK cell isolation kit. After purity evaluation, NK cells were incubated with interleukin-15 overnight. Leukemia cells were incubated in minimally toxic (20% cytotoxicity) concentrations of 6MP and MTX. The leukemia/lymphoma cells were then co-incubated with NK cells at different ratios. The NK cell-mediated leukemia/lymphoma cell cytotoxicity was measured by flow cytometric cell-mediated cytotoxicity assay, marking effector cells with lineage-specific monoclonal antibodies and staining target cells with propidium iodide and annexin-V and using microspheres for quantification of viable and apoptotic cells. The level of resistance of the respective cell sub-lines was evaluated using MTT assay. We also investigated whether NK cell exposure to same concentrations of MC drugs before co-incubation alters cytotoxicity. Surface expression of NKG2D ligands, ULBP 1, 2 and 3, MICA and MICB was studied by flow cytometry. Results: 6-mercaptopurine treatment of REH cells and MTX treatment of Raji cells resulted in enhanced NK cell-mediated elimination when compared to untreated cells by 25% and 20%, respectively. The results were similar when NK cells were exposed to the same concentrations of MC drugs before co-incubation, indicating lack of negative effect of the drug exposure in NK cells’ ability to kill. Similar experiments were conducted on resistant cells, in order to make the target cells more comparable to the residual lymphoblasts during MC. Most interestingly, the REH cells, but not the Raji cells, resistant to Ara-C and MTX showed about 14% and 4% enhancement of NK cell-mediated killing, respectively, after being exposed to the minimally toxic concentrations of MC drugs. This indicates that resistant B precursor ALL cells can be eliminated by NK cells upon MC drug exposure, but not mature B lymphoblasts, in this experimental setting. No increase in the expression of NKG2D ligands on drug treated ALL cells was observed. Conclusion: These findings suggest that enhanced susceptibility of drug-exposed leukemia cells to innate immune response may be an operational mechanism of MC. This mechanism may involve pathways other than NKG2D. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Induction of NK Cell Reactivity against B-Cell Acute Lymphoblastic Leukemia by an Fc-Optimized FLT3 Antibody

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1966 ◽  
Author(s):  
Bastian J. Schmied ◽  
Martina S. Lutz ◽  
Fabian Riegg ◽  
Latifa Zekri ◽  
Jonas S. Heitmann ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Line ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Target Cells ◽  
Dependent Manner ◽  
Cell Reactivity ◽  
Cell Acute Lymphoblastic Leukemia

Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism by which antitumor antibodies mediate therapeutic efficacy. At present, we evaluate an Fc-optimized (amino acid substitutions S239D/I332E) FLT3 antibody termed 4G8-SDIEM (FLYSYN) in patients with acute myeloid leukemia (NCT02789254). Here we studied the possibility to induce NK cell ADCC against B-cell acute lymphoblastic leukemia (B-ALL) by Fc-optimized FLT3 antibody treatment. Flow cytometric analysis confirmed that FLT3 is widely expressed on B-ALL cell lines and leukemic cells of B-ALL patients. FLT3 expression did not correlate with that of CD20, which is targeted by Rituximab, a therapeutic monoclonal antibody (mAb) employed in B-ALL treatment regimens. Our FLT3 mAb with enhanced affinity to the Fc receptor CD16a termed 4G8-SDIE potently induced NK cell reactivity against FLT3-transfectants, the B-ALL cell line SEM and primary leukemic cells of adult B-ALL patients in a target-antigen dependent manner as revealed by analyses of NK cell activation and degranulation. This was mirrored by potent 4G8-SDIE mediated NK cell ADCC in experiments with FLT3-transfectants, the cell line SEM and primary cells as target cells. Taken together, the findings presented in this study provide evidence that 4G8-SDIE may be a promising agent for the treatment of B-ALL, particularly in CD20-negative cases.

scholarly journals Exploitation of natural killer cells for the treatment of acute leukemia

Blood ◽  
2016 ◽  
Vol 127 (26) ◽  
pp. 3341-3349 ◽  
Author(s):  
Rupert Handgretinger ◽  
Peter Lang ◽  
Maya C. André
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Target Cells

Abstract Natural killer (NK) cells play an important role in surveillance and elimination of malignant cells. Their spontaneous cytotoxicity was first demonstrated in vitro against leukemia cell lines, and NK cells might play a crucial role in the therapy of leukemia. NK cell activity is controlled by an array of germ line–encoded activating and inhibitory receptors, as well as modulating coreceptors. This biologic feature can be exploited in allogeneic cell therapy, and the recognition of “missing-self” on target cells is crucial for promoting NK cell–mediated graft-versus-leukemia effects. In this regard, NK cells that express an inhibitory killer immunoglobulin-like receptor (iKIR) for which the respective major histocompatibility complex class I ligand is absent on leukemic target cells can exert alloreactivity in vitro and in vivo. Several models regarding potential donor–patient constellations have been described that have demonstrated the clinical benefit of such alloreactivity of the donor-derived NK cell system in patients with adult acute myeloid leukemia and pediatric B-cell precursor acute lymphoblastic leukemia after allogeneic stem cell transplantation. Moreover, adoptive transfer of mature allogeneic NK cells in the nontransplant or transplant setting has been shown to be safe and feasible, whereas its effectivity needs further evaluation. NK cell therapy can be further improved by optimal donor selection based on phenotypic and genotypic properties, by adoptive transfer of NK cells with ex vivo or in vivo cytokine stimulation, by the use of antibodies to induce antibody-dependent cellular cytotoxicity or to block iKIRs, or by transduction of chimeric antigen receptors.

scholarly journals Immunocytokines with Target Cell-Restricted IL-15 Activity for Induction of NK Cell Reactivity Against Acute Myeloid Leukemia (AML)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 731-731
Author(s):  
Bastian J. Schmied ◽  
Latifa Zekri ◽  
Martin Pflügler ◽  
Melanie Märklin ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Target Cell ◽  
Nk Cell ◽  
Target Antigen ◽  
Cytokine Release ◽  
Cell Reactivity ◽  
Flow Cytometric ◽  
Patent Applications ◽  
Xenotransplantation Model

Abstract Introduction: The efficacy of monoclonal antibodies (mAbs), which have substantially improved treatment options for cancer patients, largely relies on their ability to induce antibody-dependent cellular cytotoxicity (ADCC) of NK cells. Recently, we have introduced Fc-optimized (SDIE modification) antibodies targeting CD133 and CD135 (FLT3) with improved capacity to induce NK cell reactivity against AML cells (Koerner et al, Leukemia 2017; Hofmann et al, Leukemia 2012). Our FLT3 mAb termed FLYSYN is currently clinically evaluated in AML patients with minimal residual disease (NCT02789254). Notably, NK cell reactivity can be substantially increased by the cytokine IL-15, but clinical application of truly effective doses is currently prevented by substantial side effects due to unspecific immune activation (Conlon et al, JCO 2015). To overcome this limitation and to strengthen therapeutic efficacy, we fused our Fc-optimized CD133 and CD135 mAbs to an IL-15 mutant with abolished binding to IL-15 receptor α (IL-15Rα). The resulting modified immunocytokines (MIC) should substitute trans-presentation of IL-15 by binding to their target antigens on leukemic cells which facilitates stimulation of IL-15Rβ/γ on NK cells. Methods: Comparative analysis of MIC133/MIC135 binding to target cells, target antigen expression and induction of antigen shift was performed by flow cytometry using primary AML cells and target antigen transfected cell lines. NK cell activation was monitored by flow cytometric analyses of activation markers such as CD69 and CD25. Cytokine release, in particular that of IFN-γ, was measured by ELISA. Target cell killing in cocultures of healthy peripheral blood mononuclear cells (PBMC) with primary AML cells or target antigen transfected cell lines was studied by Europium, Xcelligence and flow cytometry based assays. Toxicity against healthy FLT3 expressing cells was studied by flow cytometric analysis of monocytes, dendritic cells and CD34+ cells within healthy PBMC or bone marrow. For in vivo analysis, MIC135 was tested in a NOD.Cg-Prkdc(scid)IL2rg(tmWjl)/Sz xenotransplantation model by inducing leukemia with primary AML cells and polyclonal NK cells as effector cells. Results: Functional analyses confirmed target antigen-restricted binding of MIC133/MIC135 with saturating doses reached at approximately 1µg/ml. FLT3 was found to be expressed on primary AML cells with significantly higher extent and to be less susceptible to antigen shift compared to CD133. Analysis of activation and cytokine release, the latter being particularly relevant for side effects, demonstrated that MIC proteins stimulate NK cells in a target cell-restricted manner and to a profoundly greater extent than their Fc-optimized counterparts without IL-15. In line, target cell killing induced by either MIC was clearly superior to that of the respective Fc-optimized CD133 and FLT3 mAbs as revealed by various experimental systems using primary AML cells. MIC135, which was chosen for further development due to its superior characteristics described above, did not induce unwanted effects against healthy FLT3 expressing cells and potently reduced leukemic burden in a NSG xenotransplantation model with primary AML and polyclonal NK cells. Conclusion: In summary, MIC stimulate NK cells in a target cell-restricted manner, clearly outperform Fc-optimized antibodies and thus constitute a promising treatment option for AML. Disclosures Jung: Several patent applications: Patents & Royalties: e.g. EP3064507A1. Salih:Several patent applications: Patents & Royalties: e.g. EP3064507A1.

Sign in / Sign up

Export Citation Format

Share Document